It is now well established that on most cells of the body there exists a modified, non-motile primary cilium that acts as an antenna sensing the extracellular environment, be it the lumen of a tubular organ or the extracellular matrix of connective tissues. The primary cilium ultimately transduces stimuli that result in gene expression controlling fundamental biological responses of the cell. In the past five years there has been a significant increase in the understanding to the role of the primary cilium as a sensory organelle in epithelial cells throughout the body, with the greatest breakthroughs coming from discovering certain abnormalities of the primary cilium that are linked to specific diseases, such as polycystic kidney disease in young children. [unreadable] [unreadable] For parallel advancements to be made in understanding the functional role of the primary cilium in cells of connective tissues, methodology must be developed that allows easy detection of primary ciliary incidence and orientation, in vitro and in vivo. In epithelia, the primary cilium of each cell projects into the lumen of the organ or to the surface of a monolayer culture. In this superficial position its presence and orientation can be analyzed by light microscopical techniques following experimental manipulation. Primary cilia have been described as present in chondrocytes, tenocytes, and other connective tissue cells, but experimental analysis of their function remains a significant challenge because 1) the cilium projects into the extracellular matrix, and 2) many connective tissue cells do not maintain phenotypic stability in long-term monolayer culture. The goal of this proposal is to develop rapid versatile analytical methods based on imaging using multiphoton microscopy to examine the incidence, orientation and molecular structure of the primary cilium in articular cartilage, growth plate, tendon and intervertebral disc. This methodology will put the exploration of the function of the primary cilium in connective tissues on par with that of its counterpart in epithelial tissues. [unreadable] [unreadable] Establishment of this methodology will open the potential for exploring the function of the primary cilium of connective tissue cells in vivo through analysis of the cilium 1) in mouse models with chondrodysplasias thought to be linked to abnormalities in development of cellular polarity and tissue organization; 2) during development when anisotropy of cell and matrix organization of connective tissues is being established; and 3) in experiments in living animals involving manipulation of the biomechanical environment associated with a specific joint. An ability to assess rapidly the incidence and orientation of the primary cilium will provide a methodology that currently is missing for analysis of results from in vitro experimentation at the molecular level to test the hypothesis that the primary cilium of connective tissue cells functions as a mechanosensor. [unreadable] [unreadable] [unreadable]